Single stranded RNA viruses are unique in having an RNA template which directs the biosynthesis of RNA. Upon infection of a cell, the genome of positive strand RNA viruses directs the synthesis of viral proteins which are required for the replication of the RNA. During the replication process, a negative strand RNA molecule is first produced followed by a subsequent transcription of the negative strand to produce new copies of the positive strand RNA. Richards, O. C. et al., (1990) Current Topics in Microbiol. and Immun. 161:89-119.
Despite the fact that RNA synthesis has been studied for the past thirty years or so in single stranded RNA viruses such as poliovirus, the biochemistry of single stranded RNA virus replication has not been clearly elucidated. Richards O. C. et al., 1990. Complicating the replication process is the finding that two groups of animal viruses (picornavirus and calicivirus) and five groups of plant viruses (comovirus, luteovirus, nepovirus, potyvirus, and sobemovirus) have been shown to contain a genome-linked viral protein (VPg) attached to the 5'-end of the single-stranded genome RNA. Salas, M. (1991) Annu. Rev. Biochem. 60: 39-71. See Table 3, taken from Salas, M., (1991). For example, poliovirus and encephalomyocarditis virus (EMCV), although polyadenylated at their 3' ends, are not conventionally capped at their 5' ends. Instead of having a 7-methyl guanosine triphosphate group, the RNAs are covalently linked to a VPg. Poliovirus-specific mRNA, however, isolated from infected cells lack VPg. HeLa cell extracts and rabbit reticulocyte lysates contain an enzyme activity that cleaves VPg from the 5' terminus of picornavirus RNA in vitro. It is thought that this activity modifies all newly synthesized viral RNAs destined to become mRNAs in vivo. Jang, S. K., et al., (1988) J. Virol. 62(8):2636-2643.
Picornaviridae are a large family of non-enveloped, positive-sense RNA animal viruses, comprised of six genera (Enterovirus, Parechovirus, Rhinovirus, Hepatovirus, Cardiovirus, and Aphthovirus) that contain numerous viral species (more than 200) known to cause important diseases of humans and animals, including type A viral hepatitis, aseptic meningitis, chronic heart disease and the common cold. Table 2. VPg proteins in picornaviruses are between 20 to 26 amino acids in length. Salas, 1991. In poliovirus, VPg is a protein of only 22 amino acids (Kitamura, N., et al. (1981) Nature 291, 547-553.) whose single tyrosine residue provides the link to the 5'-terminal uridylylic acid of the genome. Rothberg, P. G., et al. (1978) Proc. Natl. Acad. Sci. USA 75, 4868-4872. Ambros, V., et al. (1978) J. Biol. Chem 253, 5263-5266. Biochemical and genetic data have implicated protein(s) mapping to the 3D region of the viral polyprotein, in the formation of the O.sup.4 -(5'-uridylyl)tyrosine bond. (Takegami, T., et al. (1983) Proc. Natl. Acad. Sci. USA 80, 7447-7451; Takeda, N., et al. (1986) J. Virol. 60, 43-53; Toyoda, H., et al. (1987) J. Virol. 61, 2816-2822.
The precise role of VPg linked to the 5'-end of single stranded RNA viruses has not been clearly understood heretofore. In poliovirus, for example, the finding of VPg covalently linked to the 5'-end of poliovirus RNA, to nascent strands of poliovirus replicative intermediates, and to the poly(U) of minus strands, suggests that VPg serves as a primer for both the plus and minus strands of poliovirus RNA. Other findings suggest that a VPg precursor or a uridylylated form of VPg serves as a primer. Still another model suggests that a hairpin formed at the 3'-end of poliovirus RNA acts as a primer for minus-strand synthesis by the RNA polymerase. Salas, M. (1991).
In accordance with the present invention, it has been surprisingly found that VPg, 3D.sup.pol, poly(A) and UTP form a complex that facilitates transfer of UMP to the hydroxyl of Y3 of VPg. Transcription of a poly(A) template is then initiated at the 3' hydroxyl group of UMP (FIG. 7, arrow). Knowledge of the biochemistry of picornavirus replication allows for intervention with various compositions in order to inhibit such replication. Inhibition of replication is especially valuable in preventing and ameliorating the progression of disease caused by picornaviruses.